Development of a safe and effective HIV vaccine remains a critically important, but elusive goal. Progress towards an effective HIV vaccine will likely require a better understanding of immune effector mechanisms that can control or prevent HIV infection, as well as the derivation of immunization strategies that are significantly more potent than those currently available. The cytotoxic T cell (TCL) response is believed to play an essential role in eliminating HIV-infected cells and controlling the levels of virus replication in infected persons, and strategies for the development of HIV vaccines are increasingly focused on elicitation of strong and durable anti-HIV CTL responses. Unfortunately, newer methods to quantify CD8 T cell responses in HIV-infected persons (e.g., MHC tetramers or cytokine detection methods) do not measure lytic function, and thus may not reveal key functional differences that may exist within the population of HIV-specific CD8 T cells. While the gold standard chromium release assays for CTL cytotoxicity based on release of radioactive chromium from labeled target cells are difficult to standardize, time consuming, and inconvenient and insensitive to perform. As a result, there is a substantial need to develop new methods to quantify the lytic function of CD8 T cells in support of studies of HIV pathogenesis and vaccine evaluation. We have developed a new non-radioactive flow cytometry-based CTL assay based on caspase-mediated activation of fluorogenic substrates that is more sensitive, more convenient, and faster than available CTL assays. To refine this assay and evaluate its utility in HIV vaccine studies, particularly as conducted in developing countries, we propose to (1) to optimize and validate simple and rapid flow cytometric CTL assays, (2) to evaluate methods to optimize the detection of memory CTL responses through short term ex vivo treatment with costimulatory activators or cytokines, (3) investigate the ability of anti-HIV CTLs to kill relevant primary target cells of different hematopoetic lineages and (4) to transfer the technology of flow-cytometry based CTL assays to laboratories in developing countries where the AIDS epidemic is most acute and where major efficacy trials of candidate HIV vaccines will be conducted.